Risks and implications of  gamete and embryo freezing

By Sammy Lee

Copyright Sammy Lee, December 1999
 
INTRODUCTION

Infertility treatment is expensive, invasive and highly stressful. In vitro fertilisation (IVF) is not just about egg collection, tissue culture and embryo transfer, but includes a whole raft of procedures, necessitating many visits to the clinic. Egg collection (EC) is also a rather unpleasant procedure, which the female partner must undergo. One way of reducing exposure to EC and other unpleasant aspects of IVF is to develop strategies which maximise the benefits accruing from each procedure. Embryo freezing plays a major role in this type of  startegy.

WHAT ARE THE RISKS OF FREEZING?

No serious risks have yet been observed. It has been postulated that, because freezing may cause disruption to cellular components, freezing may give rise to anomalies, either in daughter cells, resulting in disruption to major organs during embryogenesis or  damage to the nucleus where genetic information is held, resulting in chromosomal damage, which could lead to trisomy 13 and similar conditions. One of the more outspoken experts holding this view was Professor Lord Winston, medical director of the Hammersmith Hospital Fertility Unit.

With eggs freezing has produced variable results. To date few live births have resulted from frozen eggs (thus no data on risks of freezing eggs), whilst on the other hand sperm freezing works relatively well. In the hands of experts sperm freezing yields high recovery rates on thawing. No known increase in congenital anomalies have been observed in offspring resulting from the use of frozen thawed sperm.

WHAT IS FREEZABLE?

Not every sperm, egg or embryo is freezable. A few semen samples are completely unsuitable and result in <5% motility recovered. Likewise some embryos will not thaw (50-70% of embryos will thaw intact after freezing)
Probably the same goes for eggs, but as mentionned beforehand, there is insufficient data on this matter at present. When consenting to and considering whether to have gametes or embryos frozen it is important not to have unrealistic expectations.

FREEZING AND THAWING EMBRYOS HOW IS IT DONE?

Embryos are frozen by infusing them with a special cryoprotectant. This helps to prevent ice formation inside the embryos. It is this which causes damage during freezing. Cryoprotectant must slowly enter the cells of the embryo, after which the embryos are cooled at a controlled and slow rate using a computerised automated freezer. The freezing rate is as slow as –0.1 C per minute! The embryos must undergo a special procedure at –6.5 C called “seeding”. This causes the cryoprotectant in and around the embryos to freeze suddenly, helping to reduce freezing related damage (ice crystals). After this the controlled rate freezing continues until -90 C, after which the embryos are plunged into the liquid nitrogen.

To thaw, the embryos are thawed rapidly to room temperature and then placed into a special thawing medium containing sucrose at high concentration. This prevents the cells of the embryo from swelling too quickly as the cryoprotectant is washed out. After about 10 minutes or so in thawing medium, the embryos may be slowly placed into normal culture medium and then grown for a further 24 hours in the incubator or even transferred immediately.

EGG FREEZING-CURRENT STATE OF PLAY

To date, only about 8 live births have been achieved with frozen-thawed eggs. Improvement in this technology would have a major impact on ovum donation, providing more access to this option. Furthermore, as more is learnt about in vitro maturation of oocytes, egg freezing technology will revolutionise future choice for couples and individuals regarding their reproductive options. Egg freezing seems to affect the eggs ability to be fertilised normally, but this has now been found to be overcome by using ICSI treatment.
 

OVARY/TESTIS FREEZING-CANCER OPTIONS

Cancer strikes one in three at some point in their lives. Invariably, life saving treatment poses a threat to a person`s fertility. More recently, it is becoming more routine to offer a person, ovary or testis tissue freezing prior to embarking on chemotherapy. In some cases egg freezing or semen freezing is also a consideration. Freezing ovarian or testicular tissue is similar to embryo or sperm freezing, but varies only in the additional time allowed for infusion of the cryoprotectant. Becuse there is more tissue, the  cryoprotectant needs more time to pervade all the tissue.
 

SEMEN FREEZING

Semen freezing has been done since  before the advent of  IVF. Reasons for freezing semen vary from the need to store samples for donor insemination to the need to store samples for men who are about to undergo chemotherapy for life threatening diseases. Likewise, men undergoing andropause (primary testicular failure would also benefit from semen storage). Semen freezing has also been done for absent husbands, anxious husbands (one`s who have problems producing samples) and for men with consistently variable semen analyses. Freezing more recently has been done on sperm retrieved through surgery (MicroEpididymal Sperm Aspiration/Percutaneous Epididymal Sperm Aspiration/Testicular Sperm Extraction).

HOW IS SEMEN FREEZING DONE?

Semen is mixed with cryoprotectant prior to freezing in liquid nitrogen. Cryoprotectant is made up from a buffered basal salt solution containing glycerol (10-15%), citrate and fructose amongst other salts. The cryoprotectant seeps into the sperm and helps to protect them against damage when they are frozen. The cryoprotectant is slowly added to the semen. If the initial semen volume is 2 ml, about 1.5-2 ml of cryoprotectant will be added. Once the mix is complete, it is aliquoted out into special cryotubes (usually 1ml at a time). These tubes are then suspended over the vapour phase of liquid nitrogen (-90 C)  for 30 minutes. At the end of this time the tubes are plunged directly into the liquid nitrogen. Once immersed, the samples will keep indefinitely until thawed! It has been mooted that freezing may damage the chromosomes within the sperm heads. However, these concerns have not yet been substantiated.

When needed frozen semen is thawed by removing the cryotube from the freezer and immediately placing it at 37 C in a hot block or incubator. The sample is brought very quickly to body temperature after which it is washed with fresh culture medium. After two washes, the sperm are swum up, after which the preparation is ready for IUI, IVF or ICSI.

ISSUES CONCERNING FREEZING

Earlier the idea of unrealistic expectations was mooted. Is freezing truly worthwhile? Is it value for money? Difficult questions to answer, but very relevant ones! Semen freezing costs about £ 300 generally (inclusive of one years storage). Embryo and egg freezing costs about £ 450 (also inclusive of one years storage). Thereafter there is usually a maintenance charge of £ 200 per annum. Therefore, if the sperm, eggs or embryos are of dubious quality, freezing may be of little benefit. Under such circumstances the cost benefit analysis would indicate very little chance of success despite a substantial outlay of money (upwards of £ 300-450). Provided egg, sperm and embryos show morphology in keeping with reasonable thaw survival rates, freezing is worthwhile (i.e. at least 4 in 10 eggs/embryos and 1 in 10 sperm surviving freeze and thaw). Success rates with frozen-thawed eggs/embryos and sperm also play a role in determining the benefit of freezing. Success rates vary enormously. The HFEA gives data for frozen embryo replacements (national average of about 8% per frozen-thawed embryo replacement) and for donor insemination with frozen semen (national average about 8% per cycle of insemination). If a unit is getting substantially lower success rates than the average, careful consideration is needed before deciding whether to proceed with freezing or not.

DOES THE STAGE AT WHICH AN EMBRYO IS FROZEN AFFECT SUCCESS RATES?

Freezing of embryos may be done at pronuclear (when the egg has just been fertilised and the two pronuclei (PN) are still visible), the 4-cell stage (when embryos are normally transferred) the morula stage (8-32 cell stage) or at the blastocyst stage. Many units report best results (up to 35% per frozen embryo replacement) with PN stage freezing. Perhaps because the embryos are at a specific cell cycle stage when freezing is optimal. On the down side, it is believed that often the best embryos may be frozen at this stage and that the embryos then available for transfer may be of a lower quality. Freezing at the 4-cell is most common, even today. This routinely gives success rates of 5-15% success rates (live birth rate per frozen-thwaed emrbyo replacement), a reasonably efficient and effective technique. Blastocyst freezing has historically produced live birth rates of about 8%, but with the advent of blastocyst culture, these success rates may improve substantially.

NATURAL OR STIMULATED TRANSFER CYCLES

Other factors which may determine success rates is whether the embryos are replaced in natural or manipulated cycles. There are pros and cons. Some argue natural cycles are best. Where the endometrium is always optimal in natural cycles, why bother with more “messing around”? However, where the endometrium is always sub-optimal, the use of hormones to make the environment “ideal” is a logical procedure.

THE EFFECT OF FREEZING ON SEMEN QUALITY
DOES FREEZING SEMEN AFFECT ITS FERTILITY?

Freezing has been found to affect sperm function. Frozen thawed sperm, when compared with fresh sperm from the same person show impaired mucus penetration. Furthermore, HFEA data shows that donor insemination results since 1991 have been poor (6-8%), as compared with prior use of fresh donor samples (10-12%). More recently, as units have acknowledged the detrimental effects of freezing, and started to prepare frozen-thawed donor samples, results have started to approach fresh success rates (10-15 % live birth rate per cycle) once again (i.e. the use of intrauterine insemination with washed and swim up frozen-thawed semen enhances treatment outcome). It has also been observed that IVF with frozen autologous semen (5-10% live birth rates per cycle) is much less successful than IVF with frozen donor semen (25-33% live birth rate per cycle). Thus it may be inferred that when semen quality is poor, freezing is much more detrimental than when semen quality is good as in the case of donor sperm.